Int J Clin Exp Med 2016;9(7):12976-12983
www.ijcem.com /ISSN:1940-5901/IJCEM0022021
Original Article
Dual specificity protein phosphatase 1 (DUSP1)
in normal pregnancy and preeclampsia
Zhi-Ling Yang, Hong-Mei Li, Wei Huang, Lei Han, Li-Li Yu, Li Li
Department of Obstetrics and Gynecology, Research Institute of Surgery and Daping Hospital, Third Military
Medical University, Chongqing 400042, China
Received December 16, 2015; Accepted May 5, 2016; Epub July 15, 2016; Published July 30, 2016
Abstract: Objectives: Preeclampsia is best described as a pregnancy-specific syndrome of reduced organ perfusion
secondary to vasospasm and endothelial activation. It is severe and clinically important manifestations of placental
insufficiency. Our purpose was to study DUSP1 expression in normal human pregnancy and preeclampsia. Study
design: We used ELISA, RT-PCR and Immuno-histochemistry to characterize DUSP1 gene expression and protein
levels in different ages of the first trimester pregnancy, late pregnancy and in severe preeclampsia. DUSP1 protein
was detected in venous of every patient and villi of the first trimester pregnancy, placentas of normal pregnancy
and preeclampsia pregnancy. Villi was gained from those women who terminated their pregnancy during the first
trimester and the placenta was gained from normal and preeclampsia pregnancy during the cesarean. Results:
DUSP1 protein was significantly lower in severe preeclamptic placentas compared to normal pregnancy (P<0.05).
The express of DUSP1 was varied from different ages of pregnancy. There was a trend that DUSP1 level decreased
in villi of first trimester with the gestational age increased. Conclusion: DUSP1 protein levels were significantly suppressed in severe preeclampsia. This suppression was the reason or result of preeclampsia, is still not clear. The
express of DUSP1 was varied from different ages of pregnancy. So our study demonstrated that the level of DUSP1
protein and mRNA could be a factor of PE.
Keywords: Dual specificity phosphatase 1 (DUSP1), expression, preeclamptic, preeclampsia, ELISA, RT-PCR,
immuno-histochemistry
Introduction
Dual specificity protein phosphatases are a
superfamily, including DUSP1, DUSP2, DUSP5,
DUSP9 etc, almost 30 types of DUSP [1]. All the
DUSP participate in signal transduction pathways inactivating mitogen-activated protein
kinases (MAP kinases). Researches have tested that downregulation of DUSP1 induced
changes in the expression levels of genes
involved in specific biological pathways, including angiogenesis, MAP kinase phosphatase
activity, cell-cell signaling and tyrosine-kinase
receptor activity [2]. Its role is very commonly
recognized in tumor biology. But there is little
about the relationship between DUSP1 and trophoblast and diseases. To understand more
about the involvement of DUSP1 in normal
pregnancy and preeclampsia, we performed
these tests to analyze expression of DUSP1 in
placenta of preeclampsia and normal late pregnancy and villi of the first trimester pregnancy
woman and in venous of them. And the
researchers have investigated that DUSP1 was
an important negative regulator of the acute
inflammatory response and it protected overactivation of hypoxia-inducible factor 1 (HIF-1)
through inactivating ERK MAPK [3]. Another
studies [4-8] show inflammatory response and
HIF-1α play an important role in preeclamptic
patients, so we care about how DUSP1 express
in normal and abnormal placenta, and what is
the role of DUSP1 in pregnancy and its’
complications?
Therefore we tested DUSP1 expression in normal human gestation and in severe Preeclampsia (sPE). This study was one part of our foundation, the National Natural Science Foundation of China (No. 81070505).
DUSP1 in normal pregnancy and preeclampsia
Table 1. Clinical characteristics of normal term pregnant and preeclamptic patients
Term pregnant
Preeclamptic
patients (n=44)
patients (n=26)
Age (years)
28.65±3.30
30.11±5.98
No. of primiparas
32 (72.7%)
21 (80.8%)
Systolic blood pressure (mmHg)
114.07±8.72
152.59±8.13*
Diastolic blood pressure (mmHg)
74.66±6.93
108.22±8.81*
Gestational age at delivery (weeks)
39.2±2.1
35.2±2.4*
Fetal birth weight (grams)
3379.10±346.28 2303.22±810.03*
*P<0.05 stand for the difference between the two groups was significant. The
systolic and diastolic blood pressure of preeclamptic patients was much higher than
term pregnant patients, then the gestational age at delivery and fetal birth weight of
preeclamptic patients were significantly lower than the other group.
Table 2. ELISA for DUSP1 protein levels in different group (conc:
pg/ml)
Group
n
OD 450
conc
P
The first trimester women 94 3.82±0.83 991.42±386.23
The term pregnant women 44 3.59±0.78 929.62±336.17
The preeclamptic women 26 3.19±0.90* 853.19±444.76* P<0.05
DUSP1 protein levels varied in different group. The results of the preeclamptic
women were significant lower than the other two groups. *P<0.05.
Materials and methods
Patient selection and sample (venous and placenta) collection
Between January and December 2013, 94
first trimester villous tissues between 5-10
weeks of gestation, 44 normal term gestation
and 26 women with severe preeclampsia were
admitted to the Department of Obstetrics
and Gynecology, Daping Hospital and Xi’nan
Hospital, Third Military Medical University were
included in the study. Usually, there were about
800 pregnancies every month in these two
hospital, we just get little part of them to be
the inclusion because of the donation of their
venous blood and placenta. Preeclampsia was
defined according to the criteria stated in
Williams Obstetrics (23rd edition), which included blood pressure of ≥140/90 mmHg for two
or more tests after 20 weeks’ gestation but
without previous hypertension history, In addition the diagnosis of preeclampsia required
proteinuria, more than +1 on a dipstick. All
these patients have no history of chronic hypertension, diabetes mellitus, nephropathy etc.
The samples of the first trimesters of normal
pregnancy were collected during the procedure
of induced abortion with their agreement. All
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the placental tissues were obtained by cesarean section,
which including placenta of
the normal term pregnancy
and preeclamptic women at
the third trimester. At the
same time, the cord blood of
every patient was collected
10 ml in the anticoagulation
tube. All the patients has
signed informed consent to
approve for donation of samples. Placental collection was
performed as per protocol.
Once collection was complete, the sample was sent
to the seventh Department
of Daping hospital and research institute of surgery
for analysis. Ethics approval
was granted and written informed consent was obtained from all the patients for
donation of samples of their
villi or placenta and blood
to the hospital.
The characteristics of the samples were summarized in Table 1.
Protein isolation
The villi and placenta tissues were washed
by 0.9% physiological saline three times till
clear, then dried with filter paper and weighed
by the microbalance. The weight of tissues was
between 40 mg to 60 mg. Put the tissues in
the Eppendorf Tubes/1.5 ml centrifuge Tubes.
Protein were extracted in RIPA buffer (Beyotime Institute of Biotechnology, PR China, 50
mM TrisHCl, 150 mM NaCl, 1% Triton ×100,
1% Na Deoxycholate and 0.1% SDS) containing protease inhibitor Phenylmethanesulfonyl
fluoride (PMSF, 100 mM, Beyotime Institute of
Biotechnology, PR China). Protein concentration was determined by BCA Protein Assay Kit
(Beyotime Institute of Biotechnology). All measurements were performed twice then get the
mean values. Aliquots were stored at -80°C.
ELISA for DUSP1
ELISA assay procedure was performed according to the instruction of the DUSP1 (Human)
Cell-Based ELISA Kit (96 assays, Version: 01.
Abnova). Proteins were extracted as above.
Int J Clin Exp Med 2016;9(7):12976-12983
DUSP1 in normal pregnancy and preeclampsia
Table 3. ELISA for DUSP1 protein levels in villi of the
first trimester group of different ages (conc: pg/ml)
Gestational age n
OD 450
conc
P
X≤6 weeks
19 3.87±0.72 999.36±316.16 P>0.05
6<X≤7 weeks
51 3.83±0.75 993.95±322.27
X>7 weeks
24 3.20±0.89 979.02±369.05
DUSP1 protein levels varied in different ages of the first trimester
group. But the difference was not significant (P>0.05).
Table 4. DUSP1 mRNA of RT-PCR in placenta
of the different groups (conc =2-ΔΔCt)
Group
The first trimester women
The term pregnant women
The preeclamptic women
ΔCt
1.5
5.2
7.33
DUSP1
P
0.3535
0.0263
0.0062 P<0.05
DUSP1 mRNA in placenta of the different groups varied.
The DUSP1 mRNA of the the preeclamptic women was
significant lower than the other two groups (P<0.05).
Every sample was tested twice then get the
mean values.
ELISA for DUSP1 protein levels in different
group (concentration (conc) pg/ml), Table 2.
ELISA for DUSP1 protein levels in villi of the first
tremister group (concentration (conc) pg/ml)
Table 3.
DUSP1 mRNA detection by RT-PCR
Total RNA was extracted from the three groups:
villous of the first trimester, placenta tissue of
term pregnancy and Preeclampsia group. RNA
was extracted by using the TRIsol Reagent
extraction method (as per manufacturer’s
instructions) (Trizol R0016, Beyotime Institute
of Biotechnology, China). Reverse transcription
and real-time PCR were performed on DNAse
treated RNA samples as previously described.
Primers were designed based on the nucleocapsid gene of DUSP1 by Primer Express 5.0
software. The real-time RT-PCR assay had a
detection limit of 5 copies, with a range of
detection between 5×106 -5 copies. GAPDH
was chosed to be the control. The standard
curve was prepared based on the linear relationship between the amount of plasmid DNA
and cycle threshold (Ct). Collected samples
were detected with the real-time RT-PCR assay
and every sample was tested three times, positive results were used for quantitative analysis.
Comparative CT Method was used to analyze
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the results of real-time PCR. The expression of the DUSP1 gene was normalized to
the geometric mean of the control genes
GAPDH. All data was expressed relative to
the control.
Immuno-histochemistry for DUSP1
4% Paraformaldehyde-fixed samples were
performed overnight, then in 30% sucrosebased solution overnight. Cryosections was
chosen for immuno-histochemical analysis of
DUSP1 as previously described. SABC procedure was performed with SP-9001 Histostain™Plus Kits (ZYMED, USA). Incubated overnight
with primary anti-body (rabbit anti-DUSP1,
1:200 dilution, Sigma, Canada) at 4°C. The following day, PBS washed 3 times, then with biotinylated anti-rabbit immunoglobulin (1:200)
was Incubated for 15 min at 37°C. PBS washed 3 times. S-A/HRP was Incubated for 15
min at 37°C. Then all slides were stained with
DAB (Beyotime Institute of Biotechnology) in
the same time (40 seconds). With the same
time and solutions in order to minimize variation in intensity of stain. Negative controls
included substitution of the primary anti-serum with non-immune serum to rule out nonspecific binding.
Statistical analysis
The quantitative data are presented as mean ±
SD. Means or medians were derived from duplicate values. A one-way ANOVA was used to
determine differences in DUSP1 gene expression across gestation. The percentage data
were presented in R×2 table and the comparison was analyzed by the χ2 test. The inter-group
comparison was analyzed by using one-way
ANOVA. The analysis was performed with SPSS
13.0 Version. p-value of <0.05 were considered
significant.
Results
Table 1 summarizes the clinical characteristics
of the patients. The age of the NTP group was
lower than the sPE group (28.65±3.30 and
30.11±5.98). The rate of primipara of the
two groups were slightly different, 32 (72.7%)
and 21 (80.8%). Systolic blood pressure was
significantly lower in the NTP group (114.07±
8.72 mmHg) compared to the sPE group
(152.59±8.13, P<0.05). Diastolic blood pres-
Int J Clin Exp Med 2016;9(7):12976-12983
DUSP1 in normal pregnancy and preeclampsia
Figure 1. Primers sequences used for RT-PCR. DUSP1: Forward: 5’-AGGACAACCACAAGGCAGAC-3’ 143 kb. Reverse:
3’-AAGGTAAGCAAGGCAGATGG-5’. GAPDH: Forward: 5’-GTCAAGGCTGAGAACGGGAA-3’, 158 bp Reverse: 5’-AAATGAGCCCCAGCCTTCTC-3’.
sures in the NTP group (74.66±6.93 mmHg)
was significantly lower than in the sPE group
(108.22±8.81 mmHg, P<0.05). The gestational
age of the NTP group was significantly greater
than the sPE group (39.2±1.9, and 35.2±
4.8 weeks). Birth weight in the NTP group and
the sPE groups were 3379.10±346.28 g and
2303.22±810.03 g respectively, was and was
significantly different (P<0.05). The results
such as systolic blood pressure, diastolic blood
pressure, gestational age at delivery and fetal
birth weight were significantly different between
the two groups. The numbers of primiparas of
these two groups were different, but the rates
were not significantly different.
Table 2 shows the results of DUSP1 protein
levels of ELISA protein levels in different group.
The ELISA of DUSP1 protein level of the first
trimester women group was 991.42±166.23
pg/ml, The ELISA of DUSP1 protein level of the
term pregnant women group was 929.62±
336.17 pg/ml, and sPE group was 853.19±
444.76 pg/ml. The difference between the two
groups was significant. The result was significantly different (P<0.05).
Table 3 shows ELISA for DUSP1 protein levels in
villi of the first trimester group of different ages
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(conc: pg/ml). It means protein levels of DUSP1
varied from different ages of pregnancy. When
the gestational age was less than 6 weeks, the
average conc of DUSP1 was 999.36±316.16
pg/ml, when the gestational age was more
than 7 weeks, it was 979.02±369.05 pg/ml,
when the gestational age was between 6 and 7
weeks, it was 993.95±322.27 pg/ml, the protein levels of DUSP1 decreased when the gestational age increased. But the difference was
not significant (P>0.05). Because of the size of
the species was not a big number, it need more
work in next study.
Table 4 shows the results of DUSP1 mRNA of
RT-PCR in villi and placenta of the different
groups. The result of RT-PCR of the first trimester women group was 0.3535, the term pregnant women group was 0.0263, and the sPE
group was 0.0062, the first trimester women
group was much higher than those of the term
pregnant women and sPE group. The result was
significantly different (P<0.05).
Figure 1 shows the primers sequences and the
conditions used for RT-PCR. It also shows the
plate setup, the themal profile and the amplification plots of DUSP1.
Int J Clin Exp Med 2016;9(7):12976-12983
DUSP1 in normal pregnancy and preeclampsia
Figure 2. Representative photomicrographs of DUSP1 immuno-histochemistry in the first trimester women (A), the
term pregnant women (B) and sPE patients (C). Notable increased intensity of DUSP1 staining in first trimester villis
vs placentas at term ones. DUSP1, primary antibody 1:200.
Figure 2 shows the result of the Immunohistochemistry for DUSP1 in villi and placenta
12980
of the different groups. Representative photomicrographs of DUSP1 immuno-histochemistry
Int J Clin Exp Med 2016;9(7):12976-12983
DUSP1 in normal pregnancy and preeclampsia
in the first trimester women (A), the term pregnant women (B) and sPE patients (C) in ×400
(left) and ×200 (right). Notable increased intensity of DUSP1 staining in first trimester villis vs
placentas at term ones.
Discussion
Preeclampsia can result in maternal morbidity
and mortality [9, 10]. It is a pregnancy-specific
syndrome [11]. It usually occurs after 20 weeks
of gestation and its clinical symptoms disappear after the termination of pregnancy [12].
The minimum criteria for the diagnosis of preeclampsia are hypertension plus minimal proteinuria. Significant proteinuria is defined by
24-hour urinary protein exceeding 300 mg per
24 hours, or persistent 30 mg/dL (1+ dipstick)
in random urine samples. The more severe the
hypertension or proteinuria, the more certain is
the diagnosis of preeclampsia. Similarly, abnormal laboratory findings in tests of renal, hepatic, and hematological function increase the certainty of preeclampsia. The decreased invasion
capacity of trophoblasts in the first trimester
may be crucial for the pathogenesis of preeclampsia [13, 14]. Many biochemical markers
has been reported to be correlated with this
disease, such as ET-1 (endothelin-1) [15], VEGF
[16, 17] (vascular endothelial growth factor),
PlGF [18, 19] (placental growth factor), HIF-1α
[7, 8] et al. But there was no idea which biochemical marker was the most sensible one to
predict the preeclampsia. It has been reported
that hypoxia was a major contributor to the
abnormalities seen in the preeclamptic placenta [20].
It is known that dual-specificity phosphates
(DUSPs) played a very important role in the
mitogen-activated protein kinase (MAPK) pathway [21]. A large amount of evidence demonstrated [22] that the phosphorylation and activation status of kinases in the MAPK system
had crucial impact on the outcome of downstream events that regulate cytokine production. There were at least 13 members of the
family display unique substrate specificities for
MAPKs [23, 24]. DUSP1 was an important negative regulator of p38 MAPK activity [25].
Studies showed DUSP1 was to be related to the
negative regulation of innate immunity, inflammatory responses, and signaling events downstream of p38 MAPK in mice [3, 26, 27]. DUSP1
was a potent inhibitor of MAPK activity through
12981
dephosphorylation of MAPK. Findings indicated
that p53 was a transcriptional regulator of
DUSP1 in stress responses [28]. DUSP1 was
identified as a hypoxia responsive gene, but the
roles of it in the response of hypoxia were not
clear. Researches showed that hypoxia could
induce DUSP1 expression and suppression of
DUSP1 expression facilitated the interaction
between HIF-1 alpha subunit [3]. DUSP1 may
protect overactivation of HIF-1α through inhibiting ERK kinase activity. Just because of DUSP1
was identified as a hypoxia responsive gene,
and there is some relationship between DUSP1
and HIF-1α. Hypoxia is a major contributor to
the abnormalities seen in the preeclamptic placenta. So we suggest that DUSP1 would be a
potential marker in preeclampsia. The ELISA of
DUSP1 protein level of NTP group was higher
than sPE group, and the difference between
the two groups was significant. The result of
RT-PCR of NTP group was much higher than
that of the sPE group and the difference was
significant (P<0.05). Our study showed, DUSP1
was significantly down expressed in the placenta of preeclampsia specimens in different
methods. We also found that placental DUSP1
expression was higher in early gestation than in
term gestation and a statistically significant
correlation was found between preeclampsia
and normal pregnancy (late pregnancy). But it
was very striking result that there wasn’t difference between preeclampsia and early pregnancy. Though there were more than 400 normal
pregnancies and 5 sPE pregnanies admitted
every month in our hospital, but not so many
pregnancy persons want to donate their blood
and placenta for the research, so the number
of species was not so big.
In fact, during the clinical work, we can not
keep the gestational weeks being the same
between the normal and sPE pregnancy. Because we would chose cesarean section for
the sPE group before 37 weeks, and if the
patient was severe PE, we would do the cesarean section earlier, such as 34~36 weeks. But,
if the patient was normal pregnancy, we would
chose cesarean section for them after 39
weeks. Aslo we are not sure whether the low
expression of DUSP1 in sPE was reason or
result, and what is the function and key point of
DUSP1 in placental development of preeclampsia patients, still be unknown. So there was too
much need to learn. The mechanism mediating
the significant suppression of DUSP1 in preInt J Clin Exp Med 2016;9(7):12976-12983
DUSP1 in normal pregnancy and preeclampsia
eclampsia is unclear. Hypoxia caused significant down-regulation of DUSP1 gene expression in placenta tissue. There has been previously described that DUSP9 exerts its actions
through the MAPK pathways ERK1/2 and p38.
But what is the role of DUSP1, it need more
study. So in our next study, we want to find out
DUSP1 mRNA expression in villous trophoblast
in early and term gestation and preeclampsia
patients. We also want to find out whether there
is any difference between the different times of
gestation in the first trimester and third trimester. We also want use the DUSP1 knock-out
mouse model where DUSP1 absent expression
was associated with? What is the relation of
DUSP1 and invasive capacity of trophoblasts?
We also need observing significant difference
in the clinical symptoms between the preeclampsia patients with different level DUSP1
expression. Smets et al. have suggested that
the DUSP2 may be one of the biomarkers for
the prediction of preeclampsia. How about
DUSP1? The different level of DUSP1 expression in normal early and term pregnancy and in
preeclampsia may indicate that the examination of DUSP1 may be of great value in the prediction of preeclampsia at the clinic.
[3]
[4]
[5]
[6]
[7]
[8]
Acknowledgements
This study was supported by the grant from the
National Natural Science Foundation of China
(No. 81070505).
[9]
Disclosure of conflict of interest
None.
Address correspondence to: Drs. Li Li and Li-Li
Yu, Department of Obstetrics and Gynecology,
Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing
400042, China. Tel: +86-13808339118; Fax: +86023-68767926; E-mail: [email protected] (LL);
[email protected] (YLL)
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